A command chemical triggers an innate behavior by sequential activation of multiple peptidergic ensembles

BACKGROUND: At the end of each molt, insects shed their old cuticle by performing the ecdysis sequence, an innate behavior consisting of three steps: pre-ecdysis, ecdysis, and postecdysis. Blood-borne ecdysis-triggering hormone (ETH) activates the behavioral sequence through direct actions on the central nervous system. RESULTS: To elucidate neural substrates underlying the ecdysis sequence, we identified neurons expressing ETH receptors (ETHRs) in Drosophila. Distinct ensembles of ETHR neurons express numerous neuropeptides including kinin, FMRFamides, eclosion hormone (EH), crustacean cardioactive peptide (CCAP), myoinhibitory peptides (MIP), and bursicon. Real-time imaging of intracellular calcium dynamics revealed sequential activation of these ensembles after ETH action. Specifically, FMRFamide neurons are activated during pre-ecdysis; EH, CCAP, and CCAP/MIP neurons are active prior to and during ecdysis; and activity of CCAP/MIP/bursicon neurons coincides with postecdysis. Targeted ablation of specific ETHR ensembles produces behavioral deficits consistent with their proposed roles in the behavioral sequence. CONCLUSIONS: Our findings offer novel insights into how a command chemical orchestrates an innate behavior by stepwise recruitment of central peptidergic ensembles.     

Steroid induction of a peptide hormone gene leads to orchestration of a defined behavioral sequence.

At the end of each molt, insects shed the old cuticle by performing preecdysis and ecdysis behaviors. Regulation of these centrally patterned movements involves peptide signaling between endocrine Inka cells and the CNS. In Inka cells, we have identified the cDNA and gene encoding preecdysis-triggering hormone (PETH) and ecdysis-triggering hormone (ETH), which activate these behaviors. Prior to behavioral onset, rising ecdysteroid levels induce expression of the ecdysone receptor (EcR) and ETH gene in Inka cells and evoke CNS sensitivity to PETH and ETH. Subsequent ecdysteroid decline is required for peptide release, which initiates three motor patterns in specific order: PETH triggers preecdysis I, while ETH activates preecdysis II and ecdysis. The Inka cell provides a model for linking steroid regulation of peptide hormone expression and release with activation of a defined behavioral sequence.     

Molecular cloning and biological activity of ecdysis-triggering hormones in Drosophila melanogaster

Ecdysis-triggering hormones (ETH) initiate a defined behavioral sequence leading to shedding of the insect cuticle. We have identified eth, a gene encoding peptides with ETH-like structure and biological activity in Drosophila melanogaster. The open reading frame contains three putative peptides based on canonical endopeptidase cleavage and amidation sites. Two of the predicted peptides (DrmETH1 and DrmETH2) prepared by chemical synthesis induce premature eclosion upon injection into pharate adults. The promoter region of the gene contains a direct repeat ecdysteroid response element. Identification of eth in Drosophila provides opportunities for genetic manipulation of endocrine and behavioral events underlying a stereotypic behavior.     

Rescheduling Behavioral Subunits of a Fixed Action Pattern by Genetic Manipulation of Peptidergic Signaling

The ecdysis behavioral sequence in insects is a classic fixed action pattern (FAP) initiated by hormonal signaling. Ecdysis triggering hormones (ETHs) release the FAP through direct actions on the CNS. Here we present evidence implicating two groups of central ETH receptor (ETHR) neurons in scheduling the first two steps of the FAP: kinin (aka drosokinin, leucokinin) neurons regulate pre-ecdysis behavior and CAMB neurons (CCAP, AstCC, MIP, and Bursicon) initiate the switch to ecdysis behavior. Ablation of kinin neurons or altering levels of ETH receptor (ETHR) expression in these neurons modifies timing and intensity of pre-ecdysis behavior. Cell ablation or ETHR knockdown in CAMB neurons delays the switch to ecdysis, whereas overexpression of ETHR or expression of pertussis toxin in these neurons accelerates timing of the switch. Calcium dynamics in kinin neurons are temporally aligned with pre-ecdysis behavior, whereas activity of CAMB neurons coincides with the switch from pre-ecdysis to ecdysis behavior. Activation of CCAP or CAMB neurons through temperature-sensitive TRPM8 gating is sufficient to trigger ecdysis behavior. Our findings demonstrate that kinin and CAMB neurons are direct targets of ETH and play critical roles in scheduling successive behavioral steps in the ecdysis FAP. Moreover, temporal organization of the FAP is likely a function of ETH receptor density in target neurons.     

Two subtypes of ecdysis-triggering hormone receptor in Drosophila melanogaster

Insect ecdysis is a hormonally programmed physiological sequence that enables insects to escape their old cuticle at the end of each developmental stage. The immediate events leading to ecdysis, which are initiated upon release of ecdysis-triggering hormones (ETH) into the bloodstream, include respiratory inflation and sequential stereotypic behaviors that facilitate shedding of the cuticle. Here we report that the Drosophila gene CG5911 encodes two functionally distinct subtypes of G protein-coupled receptors through alternative splicing (CG5911a and CG5911b) that respond preferentially to ecdysis-triggering hormones of flies and moths. These subtypes show differences in ligand sensitivity and specificity, suggesting that they may play separate roles in ETH signaling. At significantly higher concentrations (>100-fold), certain insect and vertebrate peptides also activate these receptors, providing evidence that CG5911 is evolutionarily related to the thyrotropin-releasing hormone and neuromedin U receptors. The ETH signaling system in insects is a vital system that provides opportunities for the construction of models for the molecular basis of stereotypic animal behavior as well as a target for the design of more sophisticated insect-selective pest control strategies.     

Knockdown of ecdysis-triggering hormone gene with a binary UAS/GAL4 RNA interference system leads to lethal ecdysis deficiency in silkworm

Ecdysis-triggering hormone (ETH) is an integration factor in the ecdysis process of most insects, including Bombyx mori (silkworm). To understand the function of the ETH gene in silkworm, we developed an effective approach to knockdown the expression of ETH in vivo based on RNA interference (RNAi) and a binary UAS/GAL4 expression system that has been successfully used in other insect species. Two kinds of transgenic silkworm were established with this method: the effector strain with the ETH RNAi sequence under the control of UAS and the activator strain with the GAL4 coding sequence under the control of Bombyx mori cytoplasmic actin3. By crossing the two strains, double-positive transgenic silkworm was obtained, and their ETH expression was found to be dramatically lower than that of each single positive transgenic parent. Severe ecdysis deficiency proved lethal to the double-positive transgenic silkworm at the stage of pharate second instar larvae, while the single positive transgenic or wild-type silkworm had normal ecdysis. This UAS/GAL4 RNAi approach provides a way to study the function of endogenous silkworm genes at different development stages.     

昆虫蜕皮行为的生理生化和分子生物学研究进展

羽化激素与蜕皮触发激素诱发昆虫蜕皮行为及蜕皮末期的其它生理变化。羽化激素在一些特定的脑神经分泌细胞中合成,在蜕皮激素的调控下,释放到中枢神经系统和血淋巴中。蜕皮触发激素是由Inka细胞分泌的,直接作用于中枢神经系统,触发前蜕皮和蜕皮行为。越来越多的证据表明羽化激素可能存在于所有的昆虫中,并作为一种调节蜕皮的一般性激素机制。     

Functional analysis of four neuropeptides, EH, ETH, CCAP and bursicon, and their receptors in adult ecdysis behavior of the red flour beetle, Tribolium castaneum

Ecdysis behavior in arthropods is driven by complex interactions among multiple neuropeptide signaling systems. To understand the roles of neuropeptides and their receptors in the red flour beetle, Tribolium castaneum, we performed systemic RNA interference (RNAi) experiments utilizing post-embryonic injections of double-stranded (ds) RNAs corresponding to ten gene products representing four different peptide signaling pathways: eclosion hormone (EH), ecdysis triggering hormone (ETH), crustacean cardioactive peptide (CCAP) and bursicon. Behavioral deficiencies and developmental arrests occurred as follows: RNAi of (1) eh or eth disrupted preecdysis behavior and prevented subsequent ecdysis behavior; (2) ccap interrupted ecdysis behavior; and (3) bursicon subunits resulted in wrinkled elytra due to incomplete wing expansion, but there was no effect on cuticle tanning or viability. RNAi of genes encoding receptors for those peptides produced phenocopies comparable to those of their respective cognate neuropeptides, except in those cases where more than one receptor was identified. The phenotypes resulting from neuropeptide RNAi in Tribolium differ substantially from phenotypes of the respective Drosophila mutants. Results from this study suggest that the functions of neuropeptidergic systems that drive innate ecdysis behavior have undergone significant changes during the evolution of arthropods.     

The Saccharomyces cerevisiae ETH1 gene, an inducible homolog of exonuclease III that provides resistance to DNA-damaging agents and limits spontaneous mutagenesis

The recently sequenced Saccharomyces cerevisiae genome was searched for a gene with homology to the gene encoding the major human AP endonuclease, a component of the highly conserved DNA base excision repair pathway. An open reading frame was found to encode a putative protein (34% identical to the Schizosaccharomyces pombe eth1(+) [open reading frame SPBC3D6.10] gene product) with a 347-residue segment homologous to the exonuclease III family of AP endonucleases. Synthesis of mRNA from ETH1 in wild-type cells was induced sixfold relative to that in untreated cells after exposure to the alkylating agent methyl methanesulfonate (MMS). To investigate the function of ETH1, deletions of the open reading frame were made in a wild-type strain and a strain deficient in the known yeast AP endonuclease encoded by APN1. eth1 strains were not more sensitive to killing by MMS, hydrogen peroxide, or phleomycin D1, whereas apn1 strains were approximately 3-fold more sensitive to MMS and approximately 10-fold more sensitive to hydrogen peroxide than was the wild type. Double-mutant strains (apn1 eth1) were approximately 15-fold more sensitive to MMS and approximately 2- to 3-fold more sensitive to hydrogen peroxide and phleomycin D1 than were apn1 strains. Elimination of ETH1 in apn1 strains also increased spontaneous mutation rates 9- or 31-fold compared to the wild type as determined by reversion to adenine or lysine prototrophy, respectively. Transformation of apn1 eth1 cells with an expression vector containing ETH1 reversed the hypersensitivity to MMS and limited the rate of spontaneous mutagenesis. Expression of ETH1 in a dut-1 xthA3 Escherichia coli strain demonstrated that the gene product functionally complements the missing AP endonuclease activity. Thus, in apn1 cells where the major AP endonuclease activity is missing, ETH1 offers an alternate capacity for repair of spontaneous or induced damage to DNA that is normally repaired by Apn1 protein.     

Signal transduction in eclosion hormone-induced secretion of ecdysis-triggering hormone

Inka cells of insect epitracheal glands (EGs) secrete preecdysis and ecdysis-triggering hormones (PETH and ETH) at the end of each developmental stage. Both peptides act in the central nervous system to evoke the ecdysis behavioral sequence, a stereotype behavior during which old cuticle is shed. Secretion of ETH is stimulated by a brain neuropeptide, eclosion hormone (EH). EH evokes accumulation of cGMP followed by release of ETH from Inka cells, and exogenous cGMP evokes secretion of ETH. The secretory responses to EH and cGMP are inhibited by the broad-spectrum kinase inhibitor staurosporine, and the response to EH is potentiated by the phosphatase inhibitor calyculin A. Staurosporine did not inhibit EH-evoked accumulation of cGMP. Changes in cytoplasmic Ca2+ in Inka cells during EH signaling were monitored via fluorescence ratioing with fura-2-loaded EGs. Cytoplasmic Ca2+ increases within 30-120 s after addition of EH to EGs, and it remains elevated for at least 10 min, corresponding with the time course of secretion. Secretion is increased in dose-dependent manner by the Ca2+-ATPase inhibitor thapsigargin, a treatment that does not elevate glandular cGMP above basal levels. The secretory response to EH is partially inhibited in glands loaded with EGTA, while cGMP levels are unaffected. These findings suggest that EH activates second messenger cascades leading to cGMP accumulation and Ca2+ mobilization and/or influx and that both pathways are required for a full secretory response. cGMP activates a staurosporine-inhibitable protein kinase. We propose that Ca2+ acts via a parallel cascade with a time course that is similar to that for cGMP activation of a cGMP-dependent protein kinase.     

Structure-activity relationship of ETH during ecdysis in the tobacco hornworm, Manduca sexta

In insects, ecdysis or shedding of the old cuticle, consists of a series of behaviors that are regulated by the coordinated actions of a number of neuropeptides, one of which is ecdysis triggering hormone (ETH). ETH acts directly on central pattern generators of the abdominal ganglia to trigger onset of pre-ecdysis behaviors, as well as indirectly to activate release of eclosion hormone, thereby inducing onset of ecdysis behaviors through a cGMP-mediated mechanism. We assessed the minimal C-terminal amino acids required for biological activity of ETH, by assessing: (i) onset of pre-ecdysis and ecdysis behaviors in vivo, after injection of peptide analogs, (ii) onset of fictive pre-ecdysis and ecdysis motor patterns in vitro, as recorded extracellularly, after incubation of the CNS with the peptide analogs, and (iii) accumulation of cGMP within cells of the abdominal ganglia, as assessed immunohistochemically. Amidation of ETH at the C-terminus was required to elicit a biological response in vivo and in vitro, as well as an accumulation of cGMP within the CNS. The five amino acid amidated C-terminus of ETH (NIPRMamide) was the minimal moiety able to induce a robust pre-ecdysis response in vivo and in vitro, while a seven amino acid core (NKNIPRMa) was required for induction of ecdysis, including accumulation of cGMP immunoreactivity within the CNS. Analogs smaller than 12 amino acids in length were only active at very high concentrations in vivo, suggesting that smaller fragments might be susceptible to hemolymph degradation. Some alanine substitutions or removal of internal amino acids altered the activity of ETH, as well as the time of onset of ecdysis behaviors, suggesting that internal amino acids play a role in maintaining proper folding of the peptide for successful binding or activity at the ETH receptor.     

Nonsense Mutation in the Regulatory Gene ETH2 Involved in Methionine Biosynthesis in SACCHAROMYCES CEREVISIAE

Ethionine-resistant mutants, mapping at the locus eth2-the product of which is involved in pleiotropic regulation of methionine biosynthesis-have been isolated in a strain carrying five ochre nonsense mutations. Selection for nonsense suppressors in such a strain led to characterization of several allele-specific but gene non-specific suppressors which are active on the recessive heteroallele eth2-2 (resulting in partial recovery of sensitivity toward ethionine) as well as on the five other suppressible alleles. Two of these suppressors are unlinked to the eth2 gene and either dominant or semi-dominant. It is concluded that the mutation eth2-2 resulted in a nonsense codon. Enzyme studies indicate that this mutation results in a complete absence of an active product of gene eth2, in contrast with the effect of a former mutation eth2-1 which was interpreted as leading to a modified product of this gene (Cherest, Surdin-Kerjan and de Robichon-Szulmajster 1971). This conclusion is based on the absence of repressibility of methionine group I enzymes and the observation that in a heteroallelic diploid, eth2-1 expression is not masked by eth2-2. The nonsense suppressors studied lead to at least partial recovery of repressibility of methionine group I enzymes. All these results support the idea that the product of gene ETH2 is an aporepressor protein.     

Methionine biosynthesis in Saccharomyces cerevisiae: Mutations at the regulatory locus ETH2

Summary Homoallelic and heteroallelic diploids involving the eth2-1, eth2-2 and eth2-7 alleles have been studied on the basis of several criteria used for the study of haploid strains: resistance towards ethionine, overproduction of either methionine or/and S-adenosylmethionine, repressibility of methionine biosynthetic enzymes. Complete recessivity of the three alleles over the wild type allele has been observed, when resistance and methionine synthesis are considered. However, with the eth2-2 allele, repressibility corresponds more to a dose effect of the ETH2 allele than to recessivity. The implications of these findings have been discussed. Results obtained for heteroallelic combinations show significant deviations from the expected values. These results have been interpreted as indicating possible interactions between two differently impaired products of gene ETH2. They render likely that the product of this gene is at least an homopolymer.     

The Splice Isoforms of the Drosophila Ecdysis Triggering Hormone Receptor Have Developmentally Distinct Roles

To grow, insects must periodically shed their exoskeletons. This process, called ecdysis, is initiated by the endocrine release of Ecdysis Trigger Hormone (ETH) and has been extensively studied as a model for understanding the hormonal control of behavior. Understanding how ETH regulates ecdysis behavior, however, has been impeded by limited knowledge of the hormone’s neuronal targets. An alternatively spliced gene encoding a G-protein-coupled receptor (ETHR) that is activated by ETH has been identified, and several lines of evidence support a role in ecdysis for its A-isoform. The function of a second ETHR isoform (ETHRB) remains unknown. Here we use the recently introduced “Trojan exon” technique to simultaneously mutate the ETHR gene and gain genetic access to the neurons that express its two isoforms. We show that ETHRA and ETHRB are expressed in largely distinct subsets of neurons and that ETHRA- but not ETHRB-expressing neurons are required for ecdysis at all developmental stages. However, both genetic and neuronal manipulations indicate an essential role for ETHRB at pupal and adult, but not larval, ecdysis. We also identify several functionally important subsets of ETHR-expressing neurons including one that coexpresses the peptide Leucokinin and regulates fluid balance to facilitate ecdysis at the pupal stage. The general strategy presented here of using a receptor gene as an entry point for genetic and neuronal manipulations should be useful in establishing patterns of functional connectivity in other hormonally regulated networks.     

Retrograde BMP signaling controls Drosophila behavior through regulation of a peptide hormone battery

Retrograde BMP signaling in neurons plays conserved roles in synaptic efficacy and subtype-specific gene expression. However, a role for retrograde BMP signaling in the behavioral output of neuronal networks has not been established. Insect development proceeds through a series of stages punctuated by ecdysis, a complex patterned behavior coordinated by a dedicated neuronal network. In Drosophila, larval ecdysis sheds the old cuticle between larval stages, and pupal ecdysis everts the head and appendages to their adult external position during metamorphosis. Here, we found that mutants of the type II BMP receptor wit exhibited a defect in the timing of larval ecdysis and in the completion of pupal ecdysis. These phenotypes largely recapitulate those previously observed upon ablation of CCAP neurons, an integral subset of the ecdysis neuronal network. Here, we establish that retrograde BMP signaling in only the efferent subset of CCAP neurons (CCAP-ENs) is required to cell-autonomously upregulate expression of the peptide hormones CCAP, Mip and Bursicon β. In wit mutants, restoration of wit exclusively in CCAP neurons significantly rescued peptide hormone expression and ecdysis phenotypes. Moreover, combinatorial restoration of peptide hormone expression in CCAP neurons in wit mutants also significantly rescued wit ecdysis phenotypes. Collectively, our data demonstrate a novel role for retrograde BMP signaling in maintaining the behavioral output of a neuronal network and uncover the underlying cellular and gene regulatory substrates.     

Use of targetable gfp-tagged neuropeptide for visualizing neuropeptide release following execution of a behavior

Previous work has shown that a transgene consisting of a fusion between the rat atrial natriuretic peptide and a green fluorescent protein reporter (ANF-gfp) is processed, localized, and released, as would be an endogenous neuropeptide when it is expressed in the nervous system of Drosophila melanogaster using the GAL4/UAS expression system. Here we have tested the utility of this targetable transgene for detecting neuropeptide release following the execution of a peptide-controlled behavior. For the behavior we used ecdysis, the behavior expressed by insects to shed their old cuticle at the end of the molt. We found that larval ecdysis was accompanied by a readily detectable reduction in gfp fluorescence from relevant secretory cells in the periphery and peptidergic neurons in the CNS. We also found that expression of the ANF-gfp products did not have detrimental effects on larval ecdysis or adult circadian rhythmicity, when the transgene was expressed in peptidergic cells that are known to control these behaviors. Finally, we used a broadly expressed GAL4 driver to show that the UAS-ANF-gfp transgene could be used to identify axons that show a reduction in gfp fluorescence following the expression of ecdysis behavior. These findings, coupled with the availability of an increasing number of strains bearing different GAL4 drivers, suggest that this transgene will be a useful tool for identifying peptidergic neurons and secretory cells (and, eventually, their secretory product) that release their peptide content during the occurrence, in the intact animal, of a developmental, physiological or behavioral process of interest.     

Ecdysis triggering hormone ensures proper timing of juvenile hormone biosynthesis in pharate adult mosquitoes

Juvenile hormones (JHs) are synthesized by the corpora allata (CA) and play a key role in insect development. A decrease of JH titer in the last instar larvae allows pupation and metamorphosis to proceed. As the anti-metamorphic role of JH comes to an end, the CA of the late pupa (or pharate adult) becomes again “competent” to synthesize JH, which would play an essential role orchestrating reproductive maturation. In the present study, we provide evidence that ecdysis triggering hormone (ETH), a key endocrine factor involved in ecdysis control, acts as an allatotropic regulator of JH biosynthesis, controlling the exact timing of CA activation in the pharate adult mosquito. Analysis of the expression of Aedes aegypti ETH receptors (AeaETHRs) revealed that they are present in the CA and the corpora cardiaca (CC), and their expression peaks 4 h before eclosion. In vitro stimulation of the pupal CA glands with ETH resulted in an increase in JH synthesis. Consistent with this finding, silencing AeaETHRs by RNA interference (RNAi) in pupa resulted in reduced JH synthesis by the CA of one day-old adult females. Stimulation with ETH resulted in increases in the activity of juvenile hormone acid methyltransferase (JHAMT), a key JH biosynthetic enzyme. Furthermore, inhibition of IP₃R-operated mobilization of endoplasmic reticulum Ca²⁺ stores prevented the ETH-dependent increases of JH biosynthesis and JHAMT activity. All together these findings provide compelling evidence that ETH acts as a regulatory peptide that ensures proper developmental timing of JH synthesis in pharate adult mosquitoes.     

Use of targetable gfp‐tagged neuropeptide for visualizing neuropeptide release following execution of a behavior

Previous work has shown that a transgene consisting of a fusion between the rat atrial natriuretic peptide and a green fluorescent protein reporter (ANF‐gfp) is processed, localized, and released, as would be an endogenous neuropeptide when it is expressed in the nervous system of Drosophila melanogaster using the GAL4/UAS expression system. Here we have tested the utility of this targetable transgene for detecting neuropeptide release following the execution of a peptide‐controlled behavior. For the behavior we used ecdysis, the behavior expressed by insects to shed their old cuticle at the end of the molt. We found that larval ecdysis was accompanied by a readily detectable reduction in gfp fluorescence from relevant secretory cells in the periphery and peptidergic neurons in the CNS. We also found that expression of the ANF‐gfp products did not have detrimental effects on larval ecdysis or adult circadian rhythmicity, when the transgene was expressed in peptidergic cells that are known to control these behaviors. Finally, we used a broadly expressed GAL4 driver to show that the UAS‐ANF‐gfp transgene could be used to identify axons that show a reduction in gfp fluorescence following the expression of ecdysis behavior. These findings, coupled with the availability of an increasing number of strains bearing different GAL4 drivers, suggest that this transgene will be a useful tool for identifying peptidergic neurons and secretory cells (and, eventually, their secretory product) that release their peptide content during the occurrence, in the intact animal, of a developmental, physiological or behavioral process of interest. © 2004 Wiley Periodicals, Inc. J Neurobiol 59: 181–191, 2004